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    New England Biolabs new england biolabs exonuclease i
    <t>DNA</t> with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli <t>ExoI.</t> The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    New England Biolabs Exonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 17023 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e coli exonuclease iii
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    E Coli Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs escherichia coli dna polymerase i
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    Escherichia Coli Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Journal: Nature protocols

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

    doi: 10.1038/s41596-018-0055-0

    Figure Lengend Snippet: Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Article Snippet: M0293L) Lambda Exonuclease (New England BioLabs, cat.no.

    Techniques:

    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Journal: PLoS ONE

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    doi: 10.1371/journal.pone.0016925

    Figure Lengend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Article Snippet: In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done.

    Techniques: Polyacrylamide Gel Electrophoresis

    Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.

    Journal: Nucleic Acids Research

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

    doi: 10.1093/nar/gkw411

    Figure Lengend Snippet: Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.

    Article Snippet: ExoI and Ssb were purchased from New England Biolabs (Ipswich, USA) and Promega (Madison, USA), respectively.

    Techniques: Agarose Gel Electrophoresis

    DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Journal: Nucleic Acids Research

    Article Title: Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

    doi: 10.1093/nar/gkj430

    Figure Lengend Snippet: DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Article Snippet: Briefly, 4 µg of each DNA sample was digested with E.coli exonuclease III (145 U; New England Biolabs) for 1 min at 37°C, 100 mM putrescine at 37°C for 30 min (Acros Organics), both exonuclease III and putrescine, or left undigested.

    Techniques: Derivative Assay, Knock-Out

    The extended G-overhang is not due to the collapse of replication fork. ( A ) No 5′ C-rich overhang was detected during the cell cycle. Genomic DNA isolated from synchronized HeLa cells were hybridized to an 18-mer G-rich probe for detecting C-rich overhangs and hybridized to an 18-mer C-rich probe for detecting G-overhangs under native conditions. To determine the hybridization signals contributed by G- or C-rich overhangs, DNA was digested with 3′ → 5′ exonuclease ExoI, which specifically removes 3′ G-overhangs, or the 5′ → 3′ exonuclease RecJ f ) before hybridization. The same gels were then denatured to determine the total telomere signal. Asyn, asynchronized HeLa cells. ( B ) Quantitation of relative amount of G-overhangs from ( A ). ( C ) Hybridization of G-rich probe to (C 3 TA 2 ) 4 oligo. The ss (C 3 TA 2 ) 4 oligo was hybridized to G-rich probe under the native condition identical to ( A ) and separated in 20% native 0.5 × TBE polyacrylamide gel.

    Journal: The EMBO Journal

    Article Title: Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection

    doi: 10.1038/emboj.2010.156

    Figure Lengend Snippet: The extended G-overhang is not due to the collapse of replication fork. ( A ) No 5′ C-rich overhang was detected during the cell cycle. Genomic DNA isolated from synchronized HeLa cells were hybridized to an 18-mer G-rich probe for detecting C-rich overhangs and hybridized to an 18-mer C-rich probe for detecting G-overhangs under native conditions. To determine the hybridization signals contributed by G- or C-rich overhangs, DNA was digested with 3′ → 5′ exonuclease ExoI, which specifically removes 3′ G-overhangs, or the 5′ → 3′ exonuclease RecJ f ) before hybridization. The same gels were then denatured to determine the total telomere signal. Asyn, asynchronized HeLa cells. ( B ) Quantitation of relative amount of G-overhangs from ( A ). ( C ) Hybridization of G-rich probe to (C 3 TA 2 ) 4 oligo. The ss (C 3 TA 2 ) 4 oligo was hybridized to G-rich probe under the native condition identical to ( A ) and separated in 20% native 0.5 × TBE polyacrylamide gel.

    Article Snippet: To remove the 3′ G-rich overhang, total DNA was treated with the 3′ → 5′ exonuclease Escherichia coli ExoI (0.3 U/μg DNA, NEB) in 15 μl buffer (10 mM HEPES pH 7.5, 100 mM LiCl, 2.5 mM MgCl2 , 5 mM CaCl2 , 20 mM β-mercaptoethanol, 0.067 μg/μl DNase-free RNaseA) at 37°C for 1 h to overnight.

    Techniques: Isolation, Hybridization, Quantitation Assay

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Journal: Nature Communications

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    doi: 10.1038/ncomms15335

    Figure Lengend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit.

    Techniques: Mutagenesis, Two Tailed Test

    Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).

    Journal: Nucleic Acids Research

    Article Title: Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations

    doi: 10.1093/nar/gkl559

    Figure Lengend Snippet: Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).

    Article Snippet: Exonuclease III (Exo III) (NEB, 0.1 U/μl) was added for 5–10 min at 37°C.

    Techniques: Translocation Assay, Incubation, Labeling, Irradiation, Nucleic Acid Electrophoresis

    Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Journal: Nucleic Acids Research

    Article Title: Attenuation of DNA charge transport by compaction into a nucleosome core particle

    doi: 10.1093/nar/gkl030

    Figure Lengend Snippet: Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Article Snippet: T4 Polynucleotide Kinase (PNK), T4 DNA Ligase (T4 Lig) and Exonuclease III (ExoIII) were purchased from New England Biolabs.

    Techniques: Footprinting, Activity Assay